The effect of

Aspergillus

fungus on the diet

of broilers in Ahwaz

Abdoallah Beatsayah

, Seyed Asghar Khazaee

, Mohammad Chenani Khalili

Ali Nemati

and Ali Ghorbani

Departemant of Veterinary, University of Shahed Chamran Ahwaz, Khuzestan

Departemant of Animal Science, Islamic Azad University Khorasgan, Isfahan

Departemant of Poultry Nutrition, Islamic Azad University Karaj, Alborz

Departemant of Animal Science, Islamic Azad University Shahrekord, Chaharmahal Bakhtiary

Department of Animal Science, Islamic Azad UniversityScience and Research Branch, Tehran, Iran

ABSTRACT

Contamination of food and livestock feed to common poisonous fungus in the air and environment is important.

Contaminated animal feed to a atoxin producing fungus leads to the disturbance in the cycle of animal health, milk

and consumers of animal products. In this study, all isolates of Aspergillus on food have been studied with the aim

of identifying and toxin-producing. Aspergillus are a group of fungus that have replication power and abundant

growth, and are known as a part of fungal  ora of most of places, and among Aspergillus, a number of species have

capability of toxin-producing and can create disease in humans and animals. In this study, 180 samples of poultry in

the city of Ahvaz were prepared and after transfer to the laboratory and culture, puri cation was performed, then the

identi cation of isolates using the valid keys of mycology was done and extracting toxin of species on the context

of corn was also performed, also determining toxin-producing of species was done in two ways of coconut agar and

TLC. The results showed in this study several species of Aspergillus, including species of flavus A. A.parasitichus,

A.ochraceus, A.niger were identi ed. Species  avus with 52 isolates (0.028) allocated the most frequency and species

A.ochraceus with 9 isolates (4.98) had the lowest frequency. Informing toxin-producing of the fungus on poultry diet

and how toxin-producing of this fungus can be helpful in the management and prevention of infection by the fungus.

KEY WORDS: POULTRY, ASPERGILLUS, DIET

272

ARTICLE INFORMATION:

*Corresponding Author: dr.ab1395@gmail.com

Received 27

Dec, 2016

Accepted after revision 2

March, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

Biosci. Biotech. Res. Comm. Special Issue No 1:272-275 (2017)

Abdoallah Beatsayah et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS THE EFFECT OF

ASPERGILLUS

FUNGUS ON THE DIET OF BROILERS IN AHWAZ 273

INTRODUCTION

Fungus are present in abundant in the air, soil and

our environment, so in the presence of humidity and

the temperature, growth and reproduction of fungi are

escalated (Ersali et al., 2008). Some of the toxins in the

environment are fungal toxins that a atoxins are con-

sidered the most important of them (Aghababaei et al.,

2012). A atoxins are among the most important myco-

toxins produced by some Aspergillus species (Aspergil-

lus  avus, Aspergillus parasiticus) and Penicillium (Pen-

icillium Pvbrvlvm) (Jamali Emam Ghedis and Moeini.,

2010).

Aspergillus is fungal infectious disease of young

poultry and chronic disease of older poultry that occurs

with breathing dif culties. Fumigatus is the cause of

Aspergillus fungus that remains in the nature by pro-

ducing spore in nature. Fungus grows and reproduces

rapidly in temperature 37 degrees and proper humidity

but the increase of humidity causes the stop of growth.

Considering its growth speed, it eliminates one-day

chicks earlier that have lower resistance. Disease course

varies depending on the age and power of poultry that

in young poultry in acute form takes one week and in

old poultry in chronic form takes a few weeks. The exist-

ence of some fungi in feed for livestock and poultry is

natural and if they not have uncontrolled growth, they

not have health risks but some fungi that are called fun-

gus of toxin-producing have particular importance.

Mycotoxins are secondary metabolites produced by

some  lamentous fungi that infect agricultural products.

They are toxic for humans and animals and causing a

signi cant reduction in product returns and economic

losses. There are widespread reports of outbreak of fun-

gal damages in different countries. The metabolites are

produced by different species under special conditions in

terms of temperature, humidity and oxygen and are not

essential for cellular activities. Consumption of foods

contaminated with mycotoxins has been associated with

a number of human poisoning, and even poisoning with

mycotoxins can sometimes cause death (Aghababaei

et al., 2012).

Peanuts, pistachios, wheat, rice, corn, almonds and

 gs are the main hosts of the fungus and are known

as the most perfect natural environment for the growth

of a atoxin-producing fungi in the world, yet, several

types of a atoxin have been detected that among them

B1, B2, G1, G have the utmost importance in the world.

Among the different types of a atoxin known, a atoxin

B1 by the International Agency for carcinogens is placed

in Group A, research on cancer and in the meantime the

toxicity and carcinogenicity of a atoxin B1 is reported

over other types, contamination of feed for livestock and

poultry to fungus, followed by it a atoxins, in addition

to economic losses jeopardizes consumers of food with

animal origin. Therefore, tracking and evaluation of

a atoxins in food and animal feed and comparison it

with standard values in order to knowledge, proposals

and measures to prevent A atoxicosis in animals and

humans is necessary. Several studies are conducted on

investigating the presence of a atoxins in feed in Iran

and many countries (Kan and Meijer, 2007).

Clinical signs of a atoxin toxicity include: autopsy

lesions, autopsy injuries, histopathological lesions in

tissue, as well as the effects created on production indi-

ces of poultry  ocks in cases of experimental and nat-

ural occurrence of A atoxicosis in broiler chickens in

worldwide is reported (Kanungo et al., 2011). The main

body attacked by the a atoxin is liver and in human

causes severe disorders of the liver occur. In animals

also it causes problems in the gut, preventing immune

function, reducing reproduction, increasing feed con-

version ef ciency, reducing the production of milk and

egg, anemia, jaundice, reducing growth. Considering the

importance of toxin-producing spices on food and diets

of poultry in order to recognize species of toxin-pro-

ducing and how their toxin-producing seems necessary.

MATERIALS AND METHODS

In order to identify isolates of Aspergillus fungus in

poultry in Ahvaz during 2015-2016, the sampling was

done in 4 active poultry farms in different parts of the

city of Ahvaz. Samples taken were formed of various

materials such as all diets used by poultry including:

starter, middle-feeding and post-feeding, corn and soy-

beans, 150 samples were separated and placed inside

plastic bags in the refrigerator at a temperature 4 ° C.

Then the potato dextrose agar medium for culturing and

isolation of fungi from sample was used. Samples were

placed for 24 to 48 hours in incubation at 25 ° C then for

purifying fungus, new colonies grown were cultivated.

A. Puri cation of fungus

In order to purify a fungal isolates, single spore method

was used. So that by Anas  ne needle, a small amount

of Fungi spore was transferred to tube containing

distilled water and then by Lam Thomas, its dilution was

calculated. After preparing a suitable dilution, diameters

of the suspension was transferred to medium of water

agar 2% and by passing 12 to 24 hours, the colonies

grown of fungus on medium (PDA) that already poured

and cooled in the three spots was transferred and for 5

to 7 days at 25 ° C were reared (

Lanyasunya et al., 2005).

B. Identi cation of fungus

To identify fungus, different culture media was used,

including the culture of potato, dextrose, agar (PDA) is

Abdoallah Beatsayah et al.

274 THE EFFECT OF

ASPERGILLUS

FUNGUS ON THE DIET OF BROILERS IN AHWAZ BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Table 1. The sample and the number of food rations contaminated with Aspergillus fungus

Sample

Total

sample

number and percent of sample

A Flavus.A. Parasitichu.A. Niger.A. ochraceus

Corn30202020-

Soybean301257-

Starter308---

Middle-feeding305--4

Post-feeding303-5-

Context3046105

Total305231429

Percent of contamination300.0280.01822.254.98

known a public culture medium and used for the cul-

tivation and primary isolation of isolates, as well as to

identify the isolates, speci c culture mediums such as

CY20S (Czapek Yeast Extract Agar with20 sucrose) were

used.

C. Preparing context of corn

Value of 100 grams of corn separately was poured in

separate Erlenmeyer and the ratio of 100 to 27 was

added to each of the contexts of distilled water and in

temperature of 121 ° C. and 1.5 atmospheric pressure for

15 minutes was sterilized in an autoclave and 24 hours

later was autoclaved again to all the external factors to

be eliminated.

C. Inoculum of fungus to contexts of culture

After puri cation fungus, the fungus was developed on

PDA medium and then spore suspension was formed in

distilled water and 10 cc of this suspension in sterile

conditions under the hood of Laminar was added to

context and  asks with treatments were placed at room

temperature and the conditions 24 hours of light and

darkness.

D. Extraction of toxin

In order to extract Mycotoxins,  rst the contents of the

 asks for 24 hours at 73 ° C were placed and then milled

samples contaminated seeds acetonitrile - methanol

was added and then was placed for 3 hours on a shaker.

The resulting mixture was smooth with Whatman  lter

paper and extract was collected in clean Falcons and

extract was passed from puri cation column containing

a cationic resin and alumina-Carbon mix. For this

purpose, the beginning and the end of the column with

a layer of glass wool is blocked and a gram of mixture

of Almunia - carbon is added on it and again a layer

of glass wool blocked is placed on it and then cation

resin that already for 1 hour is placed in distilled water

is poured in column and then extract of samples were

passed through the column.

Passed extract of  rst column using the second puri-

 cation column includes respectively glass wool and

alumina-carbon and net glass wool and then extract

passed through the second puri cation column for sol-

vent evaporation was transferred to oven 70 °. After

drying the extract, methanol-water solution is added to

it and for an hour is placed at room temperature, then

methanol-water and acetonitrile-methanol solution was

added and in order to evaporate, the solvent again is

placed in an oven at 73 ° and  nally methanol-water

was added to dried extract and the extracts were stored

at -20 ° C (Rahimi et al., 2011).

E. Investigating toxin-producing by T LC

After extracting the toxins, solutions of sample and

a atoxin standard on TLC of TLC aluminum plate with

silica gel in a direction were dotted, after the plate was

placed in TLC tank and methanol: acetonitrile (88:12)

risen and the plate is washed up. Plate is dried in the

air and under the lamp UV (365 nm) will be judged and

blue  uorescence intensity is compared with standard

 uorescence (

Rahimi et al., 2008).

RESULTS AND DISCUSSION

The results of contamination of food rations including

corn, soybean, starter, middle-feeding, post-feeding and

context of poultry to Aspergillus fungus are in Table

1. The results showed in this study several species of

Aspergillus, including species A.  avus A. parasitichus,

A.ochraceus, A.niger were identi ed. Species of  avus

with 52 isolates (0.028) allocated the most frequency and

the species A.ochraceus with 9 isolates (4.98) had the

lowest frequency. The presence of fungi such as A. avus

with the most frequency among Aspergillus species can

be important in terms of toxin-producing; this fungus

can produce a atoxin B1 that has the highest toxicity

than other A atoxin produced by Aspergillus. As well

as there are species of other toxin-producing on diet of

poultry that including them can be noted to Aspergillus

Abdoallah Beatsayah et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS THE EFFECT OF

ASPERGILLUS

FUNGUS ON THE DIET OF BROILERS IN AHWAZ 275

parasiticus fungi. The species has the ability to produce

each 4 kinds of a atoxin B1, B2, G1, G2, and in this

sense can be considered a dangerous toxin-producing

fungus.

Ochratoxin A is one of mycotoxins produced by the

fungi Aspergillus and Penicillium. Spore of this fungus

are widely dispersed in the environment and a lot of food,

especially cereals are infected these fungus (Thompson

and Henke. 2000) hence the toxin-producing spices in

poultry as a pathogen agent could jeopardize poultry

health. Since the existence of the toxin-producing spices

on the dietary food for poultry and food can be danger-

ous, so the availability of toxin-producing agents and

how toxin-producing is very important, Aspergillus is

the cause of A atoxicosis in poultry as well as carcino-

genic in human (

Kanungo et al., 2011).

Brugfer et al (2003) in Australia on wheat and wheat

 our used in animal feed of farms studied isolated and

identi ed molds of Aspergillus, Cladosporydyom and

Penicillium, the type of fungi identi ed in this study

con rms the results of previous studies. Among the feed

of livestock, corn silage and concentrate, respectively,

had the highest number of fungal colonies. In a study

that was done in Mazandaran, corn has the highest

percentage to fungus Aspergillus  avus (

Rahimi et al.,

2008). This study also has identi ed corn silage as a

feed contaminated with Aspergillus  avus that hence,

con rms previous studies, but in this study, the most

frequency of this type of fungus has been observed in

straw and this could be due to difference in preparation

and storage conditions of animal feeds in different farms

and cities.

CONCLUSION

In our country, according to the climatic variation, kinds

of toxin-producing strains of aspergillus can contami-

nate food and cause huge physical and  nancial losses.

Also, due to diverse conditions of weather, there is the

possibility of a wide range of fungi pruducing myco-

toxins with toxins relevant in the environment, and it

should be done sanitation and prevention principles as

well as compliance with international standards for the

maintenance and storage of goods and food rations.

REFERENCES

Aghababaei, M., F. Fakhrzadegan., and A. Yazdani. 2012. Eval-

uation a atoxin contamination of animal feed, the number of

farms in the summer of 90 by ELISA. In National Congress

of Veterinary Laboratory Sciences. Semnan, Iran. (In Persian).

Berghofer, L. K., A. D. Hocking., D. Miskelly., and E. Jansson.

2003. Microbiology of wheat and  our milling in Australia.

International Journal of Food Microbiology, 85 (1): 137-149.

corn and its associated risks to wildlife species. Journal of

Wildlife Diseases, 36 (1): 172-179.

Ersali, A., R. Ghasemi., and F. B. A. Baigi. 2008. Transmission

of A atoxins from Animal Feeds to Raw and Pasteurized Milk

in Shiraz City and its Suburbs. Journal of Shaheed Sadoughi

University of Medical Sciences and Health Services, 17 (3):

175-183. (In Persian).

Jamali Emam Ghedis, N., and M. M. Moeini. 2010. A atoxin

contamination occurrence in milk and feed in Kermanshah

dairy farms by ElISA technique. Veterinary Journal (Pajouhesh

& Sazandegi), 87: 25-31. (In Persian with English abstract).

Kan, C., and G. Meijer. 2007. The risk of contamination of food

with toxic substances present in animal feed. Animal feed sci-

ence and technology, 133 (1): 84-108.

Kanungo, L., S. Pal., and S. Bhand. 2011. Miniaturised hybrid

immunoassay for high sensitivity analysis of a atoxin M1 in

milk. Biosensors and Bioelectronics, 26 (5): 2601-2606.

Lanyasunya, T., L. Wamae., H. Musa., O. Olowofeso., and I.

Lokwaleput. 2005. The risk of mycotoxins contamination of

dairy feed and milk on smallholder dairy farms in Kenya. Paki-

stan Journal of Nutrition, 4 (3): 162-169.

Rahimi, E., A. R. Kargar., and F. Zamani. 2008. Assessment of

a atoxin B levels in animal feed of dairy farms in Chaharma-

hal & Bakhtiari. Pajouhesh & Sazandegi, 79: 66-71. (In Persian

with English abstract).

Rahimi, E., M. Jafarian Dehkordi., and A. Iranpoor. 2011.

A Survey of A atoxin M1 Contamination in Iranian White

Cheese. Food Technology & Nutrition, 8 (4): 51-57. (In Persian

with English abstract).

Thompson, C., and S. E. Henke. 2000. Effect of climate and

type of storage container on a atoxin production in